[RASMB] problem with fitting

Thu Mar 20 11:37:05 PDT 2008

Hi Christopher,

just as a reminder, as you noted, SEDFIT consistently calculates the 
distributions in experimental s-values.  There are utility functions to 
convert it to s20w on the request of the user (for example the Mw button 
appearing over each peak after automatic integration will prompt a report 
among other things like mass and shape estimates also the s20w value).

The reason why it does experimental s-values is that in most cases with 
c(s) you will be able to resolve more than one species.  Since the 
transformation to s20w includes a buoyancy correction term, there would 
really need to be more than one vbar value to do the correction (... one 
for each peak...) SEDFIT leaves it up to you to identify which c(s) peak 
you're interested in and assign the vbar value accordingly.   Otherwise if 
you assume a single vbar value for all species in the distribution, some of 
the s-values may be off.  That would be a waste since the experimental 
s-values are the most precisely determined quantities (after the 
concentrations).

Peter

At 12:29 PM 3/20/2008, Chin, Christopher C. wrote:
>Hello Marina,
>
>As I try to clean up my old e-mail and notice that I miss this discussion 
>about two weeks ago.  I remember I did an experiment when Sedfit was 
>release. The reason for doing this experiment is when I analyzed the 
>sample with glycerol presence in the buffer, the S value is always smaller 
>than when I analysis the data using DCDT +, even when I put in the correct 
>density and viscosity value. So I derived an experiment using CRP protein 
>(a dimmer protein with molecular weight about 47KDa) in the presence of 
>different concentration of glycerol. I am sending this in the attachment. 
>If I use the correction constant, then your protein of 6.23S should really 
>be 1.21X 6.23=7.54 S. This will reconfirm it is a trimmer but will not 
>change the trimmer to hexamer as you expected. I hope this information is 
>useful for you.
>
>All the best,
>
>Christopher Chin
>
>
>-----Original Message-----
>From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] 
>On Behalf Of Thomas Jowitt
>Sent: Monday, March 03, 2008 4:39 AM
>To: thomas.a.jowitt at manchester.ac.uk; Fasolini, Marina [Nervianoms]; 
>rasmb at rasmb.bbri.org
>Subject: RE: [RASMB] problem with fitting
>
>Hi Marina
>
>Just a follow up to my previous reply. I notice you are running the sample 
>in 5% glycerol? The viscosity of this alone is 1.127E-2, not what you have 
>mentioned in your mail. If this is the case then this would alter the 
>S20,w of your protein substantially.
>
>Thanks
>
>Tom
>
>
>----------
>From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] 
>On Behalf Of Thomas Jowitt
>Sent: 03 March 2008 10:17
>To: Fasolini, Marina [Nervianoms]; rasmb at rasmb.bbri.org
>Subject: RE: [RASMB] problem with fitting
>
>Dear Marina
>
>First of all, unless your gel filtration is linked to a light scattering 
>module to estimate the absolute molecular mass, then I wouldn't trust the 
>estimated molecular weight from a calibrated column. A sedimentation value 
>of 6.23 is most likely to be around 150kDa unless the molecule is 
>substantially elongated, as a hexameric molecule of this size and 
>sedimentation value would have a frictional ratio of around 2.5 (certainly 
>not impossible).
>
>The two experiments that I would perform to answer these questions are, 
>firstly an equilibrium experiment to establish the correct molecular 
>weight, done with at-least three concentrations to establish if 
>oligomerisation is concentration dependant. Secondly, I would run the 
>crystal structure through a modeling program such as SOMO to generate a 
>bead model, or hydropro to create a shell around the crystal structure. 
>Either of these would give you a decent estimate of the sedimentation 
>value expected for a rigid molecule of that size.
>
>Thanks
>
>Tom Jowitt
>
>
>
>----------
>From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] 
>On Behalf Of Fasolini, Marina [Nervianoms]
>Sent: 03 March 2008 09:10
>To: rasmb at rasmb.bbri.org
>Subject: [RASMB] problem with fitting
>
>Dear AUC users,
>I have a problem in the fitting of some data and I would like to have your 
>opinion.
>
>I want to control the sedimentation of a protein in order to define the 
>state of oligomerization. It is published as forming an  hexameric homo 
>oligomer. From the crystal structure, it is a wheel like structure of 160 
>angstrom in diameter, with a central role of 15 angstrom in diameter. The 
>thickness is 20-40 angstrom. How can I expect it to sediment? How can I 
>improve my parameters or model in order to improve the fitting?
>
>The protein is 50KDa  but in gel filtration it cames out as  hexamer (300 
>KDa). In my sedimentation experiment I see one peak of  6.23S which 
>corresponds to 140KDa. Fitting of the sedimentation data was done with 
>SedFit. Do you think I can say that it is a trimer? I would expect it as 
>an hexamer.
>
>I hope someone can help me.
>
>the conditions are :
>buffer 20mM Tris pH7, 150mM NaCl, 1mM DTT, 5%gly
>Vbar 0.7397
>Viscosity 1.02530
>Density 0.01183
>
>Thanks a lot
>Marina
>
>
>
>MARINA FASOLINI
>Structural Chemistry
><http://www.nervianoms.com/>Nerviano Medical Sciences
>Viale Pasteur 10
>20014 Nerviano - Milano
><mailto:marina.fasolini at nervianoms.com>marina.fasolini at nervianoms.com
>Tel. +390331581462
>Fax. +390331581360
>
>
>_______________________________________________
>RASMB mailing list
>RASMB at rasmb.bbri.org
>http://rasmb.bbri.org/mailman/listinfo/rasmb
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20080320/e17f59b6/attachment.htm>